N, 2-carboalkoxyethyl derivatives of alpha-amino acids and of acylated alpha-amino acids



Patented Aug. 19, 1952 UNITED STATES PATENT" OFFICE N,2-CARBOALKOXYETHYL DERIVATIVES OF ALPHA-AMINO ACIDS AND OF ACYLATED" ALPHA-AMINO ACIDS Leonard L. McKinney, Eugene H. Uhing, Eugene A.-Setzkorn, and John C. Cowan, Peoria, 111., assignors to the United States of America as represented by the Secretary of Agriculture 13 Claims. (01. zed-471) (Granted under the act of March 3, 1883, as

amended April so, 1928; 370 0. G. 757) 1 a 2. This application is made under the act of March 3, 1883, as amended by the act of April R OH CO ORI 30, 1928, and the invention herein described, if patented in any country, may be manufactured The new Compounds a high boiling l qu and used'by or forthegovemment of theUnited 5 useful as intermediates in the production of States f America e governmental p s pharmaceuticals,synthetic resins, artificial fibers, throughout the world Without the nt to us plastics and insecticides, and as plasticizers.

of any reya1ty thereon I These compounds are produced from the cyano- This inventionrelates to the N,2-oarboalkoxyt y e amino acids described i pat apethylderivatives of alpha-amino acids and to a Plication iser- 34,257, fi d March 9, 1949 method for the preparation of these compounds. (110W Patent O y h taneous The invention has for its objects the provision Y Q Y iS and e fi o e aICOhOIY'SiS, of of these derivatives as anew class of chemical h n l roup compounds, and moreover their preparation from general, lc ysis iscarrie'd out by reactcyanoethyl derivatives of amino acids. g y l y ed ammo acids with an ali- The products of t invention have t folphatic alcohol n the presence of a non-oxidizing lowing general tr t m f r 1 I mineral acidfand adding sufi'icient alkali to the reaction mixture to convert the resulting salt of N- H H CO-OR Z C 2-6 F V the ester to the free ester. The reaction for ROH-C OO producing type I compounds may be represented where R is the residual group of the alphaschematically as follows:

NH-HC1 NH-CH2OH2CN ROH H HClNHCHz-CHzCOR' R-CH-COOH 7 H01 Cold RCHG o- 0 R amino acids in any of their optical isomeric'or Compounds of type II are; produced by a simiracemic forms, R represents the aliphatic radical lar process, employing v the corresponding diof thealiphatic alcohols, i. e., R'OH, and-Z is a cyanoethylated amino acids.

' member of the group consisting of H, R"OO and In the above equations other non-oxidizing CHzCHzOOOR in which R" may be an aliphatic 5 mineral acids, such as sulfuric and phosphoric or an' aromatic hydrocarbon radical. R and -Z or-the like may be substituted for thehydrogen may be combined in a single carbonto-carb0n chloride in the initial step, We prefer to em- .chain to form closed rings, such as are found ploy dry hydrogen chloride because of the ease .for example in proline and glutiminic acid. ii of removing excess acid by evaporation after Three general types of compounds are 'thus completion of alcoholysis of the nitrile group.. includedin the invention, as follows: The esterification reaction may be followed by filtering ofi the ammonium salt of the-mineral acidand weighing the quantity obtained.- Y

R-C'lH-C O0R v In ;the structural formulae given above, R om-om-co-QR may represent an aliphatic or araliphatic group.

It may also be heterocyclic, asfor example in the I N,carboalkoxyethyl derivatives. of. tryptophane CHPCHPC 0 OR and histidine; The-amino acids used in the RCH-CO 0R/ process ofthe invention may be naturally ccand curring or synthetically produced alpha-amino acids, such as for example, protein hydrolysates. 1

The amino acids or mixtures of amino acids are readily cyanoethylated in accordance with the process described in patent application 'Ser. *No.

derivatives serve as starting materials to prepare 84,257, filed March 29, 1949, and the cyanoet-hyl the compounds described in this invention.

Acylation of type I compounds may-be carried out by conventional methods using organic aoylating agents, such as with acetic acid-acetic'ane hydride, stearyl chloride, benzoyl chloride Orthe Moreover, in further reaction to form polymeric derivatives', their stability increases the overall emciency of the reaction. 1

The compounds of this invention'are di'stillable liquids, and when mixtures of amino acids, such as for example, protein hydrolysates or byproduct mixturesffrom' the manufacture of monosodium glutamate are used, the esterified compounds are readily separated by fractional distillation. This method of separation is more economical than that'of separating the free amino acids.

' The following specific examples are illustrative of the invention.

EXAMPLE 1 Eth'yl-N- (carbethoxymethyl) -bet'd-alanine, its hydrochloride and N-acetyl derivative-A threenecked round bottom flask was equipped with a stirrer, thermometer, and attached to a hydrogen chloride generator. One mole (128.1 g.) of N-(2- cyanoethyl) -glycine was placed" in the flask and 750 ml. of 99 percent ethyl alcohol was added. The flaskwas placed in an ice bath and the stirrer was started. Dry hydrogen chloride gas was bubbled into the mixtura while keeping the temperature at approximately 10C., until the increase in weight was 208 grams. T-heclear solution was then refluxed for one hour while stirring. During the refluxing ammonium chloride formed and'precipi-tated. The volume was then reduced by evaporating, at reduced pressure, toremove excess hydrogen chloride. The ammonium chloride was filtered off and washed with a small volume ofabsolute ethyl alcohol (wt.:55.0 g.-: calcd. 53.46 g. lfi anal: Found 25.62; calcd. 26.20). The clear filtrate was then divided into two equal portions.

, 7 Esther hydrochloride-The volume of the first portion was adjusted to 700 ml. with absolute ethyl alcohol and 400: ml. of ether was added. Crystallization occurred on"cooling.with Dry Ice, yielding 92g. of crystals (87 percent). Tengrams were recrystallized from 100 ml. ofalcohol and gave a melting point of 85-87 C. N5 anal: Found 5.86; calcd'.5.85;

Free esters-To the second portion of'theifiltra-te sodium bicarbonate (0.5 mole) was added and the mixture washeated on a steam bath, while: stirring, until eifervescence had ceased.

After filtering, the process wasv repeated with 0.25 moleslof sodium bicarbonate. The alcohol was then distilled ofi under reduced'p'ressure leaving an oil weighing 94.5 g. The oil was distilled at1-2 pressure and the main fraction 77.5

distilled:a:t95.-103 C. This fraction was then redistilled and 71 g. distilling at 97-100 was collected; f

N anaL i Found 6.82; calcd. 6.88 Sp. gr. 30/30 1'.1.0533

Refractive index (t:25) 1.4365

B. P. at atmos. pressure: 253-255 C. with decomposition Analysisagreed with the following formula:

Acetyl derivative of free ester.-50.82 g. (0.25 mole) of the free ester' was dissolvedin 145.5 g. (0.25 mole) of glacial acetic acidand g. (0.5 mole) of acetic anhydride was added. The reaction-flask became warm upon-addition of the anhydride and after standing overnight was warmed to 50 C. for 2 hours. The acetic acid and excess anhydride was distilled off underreduced pressure, and the residual oil was distilled 'at-l-Z mm; pressure. The main fraction distilledat 140-155 0., and was redistilled to give 58- g. (94- percent yield) or the purified p q ct I I new; Round 5.s9;ca1cu,5;71' Sp. gr. 30/30: 1.111? f Refractive index (t :25): 1.4551 B. P. at atoms, press: 305-310 C. with slight decomposition V v Analysis agreed with the following formula:

Other acylating agents, such as stearyl chloride and benzoyl chloride, may be substituted in the above example instead of the acetylating mixture to produce the corresponding stearyl and benzoyl derivatives.

EXAMPLE 2 N -bis- (Z-carbethorgethyl) -glycine-ethyZ-ester- -One-half mole (90.5 g.) of N-bis-(2-cyanoethyD-glycine was treated with alcoholic-hydrogen chloride as described in Example 1. 500 ml. of percent ethyl alcohol and 143 g. of hydrogen chloride gas was used. After refluxing for 2 hours on a steam bath, 50 g. (calcd. 53 g.) of ammonium chloride was filtered oil. After evaporating to remove excess hydrogen chloride, g. ofsodiurn-biearbonate was added and the mix-- ture war-med,;with stirring for 30 minutes. After fllteringrlihe neutralization step was repeated. Alcohol was distilled on; to give an 86 percent yield of;- the crude product. Upon distilling'at 1-2:mm. pressure, the main .;fraction came over at. 137L150 C. Upon redistilling, the main fr'ae' tion came overat;1 30"--135 C. The product was slightly cloudy; anclwwas Washedwith water and dried with calcium sulfiate togive a clear liquid.- Overall yield:, 50 percent.

N anal: Found 4.60; calcd. 4.61 Analysis agreed with the following formula:

CEz-CHgCO-OC2H5 I I ,EXAMPLE. 3 I

N (2 cdrbethoryethyl) -alpha-aZanine-ethylester, its hydrochloride [and acetyl derioative One mole (142.15 g.) of N-(Z-byanoethyD-alphaalanine was treated with 750 ml. of 99 percent ethyl alcohol and 220 g. of dry hydrogen chloride as described in Example 1. The solution'slowly cleared upon adding the first 100 g. of H01 gas. After 150 g. of HCl had been added, the solution became milky. The mixture was refiuxed on a steam ,bath for one hour and rapid precipitation of ammonium chloride was observed. The am monium chloride (50 g.; calcd. 53.5 g., N anal.: Found 25.85; calcd. 26.20) was filtered off and the filtrate was evaporated to approximately 600 ml. and filtered to remove a small amountfof arrimonium chloride that had precipitated. Thefiltrate. was adjusted to 650 ml.

I Lster hydrochloride- O 'eT-fo'urth of the' filtrate' was evaporated to dryness under reducedpre'ss ure. Benzene was added to the sirupy residue and evaporated off to remove tracesof moisture. The semi-solid residue was dissolved in fchloroform and ether was added. Upon cooling'ion Dry Ice a colorless sir-up layered out. The fiask was then set at room temperature whereupon crystallization occurred. The crystals (45 g.) were filtered off, a 71 percent yield.

N anal.: Found 5.55; calcd. 5.55 M. P.: 74-76 C.

Free ester.The remaining three-fourths of Sp. r. 30 30: 1.0222 7 I Refractive index (t'=25)i1.4325" B. P. atmos. pressure: 245 C. with "slight lzlec m position=.. Analysis agreed with the formula NHLOHLCHFG 6-0 02115 I, dcng -cnj-ooeocun Aee'tyZ derz'oative.+One-fifth mole of the free-. ester was di'ssolvedin 0.2 ,mole of glacial acetic acid and 0.4 moleof acetica'nhydride added as described, in Example 1. After distilling 'off acetic a'cid'andf'excess anhydride; the residual colorless oil was distilled at 1-2 mm. pressure and the fraction boiling at 170-175 C. was collected.

This fraction was redistilled to give a main frac-' tion of '50 g. (97 percent yield). I

Nanak. Found 539i calc*d..5.40 Sp'.:-gr..30/30.:..1.0914 I Refractive index (13:25") 1.4549,;

B. P; atmos. pressure: 304 (lwithgvery slight :brownin -.N -.(2-i'carbethoxyethyl) Zeucine,-et,hyZ-e ster..- Three-tenths mole (56 .g.) of N-(2-.-cyanoethyl)4 L-leucine was .treated with. alcoho1ic-HCl -.as

described in Example 1.. Because of. theglow solubility of the product, 700 ml. of;99,perce nt ethyl alcohol was used and a tOtalQfJl112 g- Di,

dry hydrogen chloride was added. After refluxing for 2 hours, 15.2 g. of ammoniumzchloride was filtered off (calcd. 16.05). The clear solution was then evaporated to 400 ml. and treated withone-mole of sodium bicarbonate." After filtering,-

a second treatment was made with 0.55 molesof- NaHCOs. :The alcohol was distilled under. reduced pressure leaving a residual colorless oilweighing 72 g. The oil was twice distilled and 62 g., boiling at 105-110 C. at 2 mm. pressure was obtained percent yield).

N anal.: Found 5.33; calcd. 5.40 Sp. gr. 30/30; 0.9794 Refractive index (t=25) 1.4358

B. P. atmos. pressure: 270 0'.

Analysis agreed with the formula:

0E3 NH-OH -CHCOOC1H5 cHoH2- :H-co oc2H5 CH3 I I I EXAMPLE 5 N -(2 carbethoxyethybmethionine ethylester, and its acetyl derivative.One-fourth mole (50.5 g.) of N-(2-cyanoethyl-DL-methionine was treated with alcoholic-H01 as described in Ex.-

ample 1. 350 ml. of 99 percent ethyl alcoholand 134 g. of dry I-IC'l gas was used. The reaction mixture was heated at 60-70 C. forl hour. and

13.25 g.:of NHiCl filtered off (calcd. 13.37 g.)-. The filtrate was neutralized to pH 6.9. with NaHCOa, filtered, and the alcohol distilled oil. The main fraction distilled at 145-150 C. at 1-2 mm. pressure. Yield: 70 percent.-

standing, whereas the acetyl derivative did not change on aging.

EXAMPLE 6 1 I "N '-('2-'carbethoryethyl) phenylalanine-ethyl ester-hydrochloride and the acetyl derivative of the I free ester.-N- (2 -cyanoethyl) DL-phenylalanine (0.053'mole) was treated. with 99 percent ethyl alcohol ml.) and'dry H01 (100 g.) as

described in Example 1. The ammonium chloride (2.6 g.) filtered off and the filtrate divided into equal parts:

The hydrochloride.''One-ha1f of the filtrate was placed on DryIce to effect crystallization. The crystals (6.2"g.,73 percent yield) were 'removed (N anal.: Found 4.50; calcd. 4.25);- In order to remove the small amount of NHiCL'the product was recrystallized from chloroform* ether solution. I I Y .1 N analQz Penman; calcd.'f4'.25' I n oif or o.

N anal: Found 4.1 5; calcd. 4.18 Refractive index (t=25): 1.500

Analysis agreed with'the formula:

v CHs-C oTNwHi-c'rirc 0,- Q2115 EXAMPLE 7 N-(Z carbethoxyethyl) tyrosine ethyl-ester.-Onefourth mole (58.56 g.) of N-( 2-cyanoethyl) -L-tyrosine was treated with 500 ml. of 99 percent ethyl alcohol and 163 g. of dry I-ICl as described in Example 1. After filtering oiT NH-iCl (14. g.; calcd. 13.4 g.), excess I-ICl wasrernoved byevaporating. The solution was then neutralized to. pH 6.8 with a Nal-ICOa, filtered, and the remainder of alcohol filtered off. Adar}; viscous oil was obtained in 80 percent yield. Analysis of the crude product indicated that it was suffi: ciently. pure formany uses-TN: Found 4.45; calcd. 4.53, agreeing with the formula:

EXAMPLE 8 N-(Z-cafbethoxyethyl) diethylaspartate and its acetylderivative-One-fourth mole (46.5 g.) of .N-(Z-cyanoethyl) -DL-aspartic acid. was treat-. ed with 350 ml. of 99 percent ethyl alcohol and 143 g. of dry'I-ICl as described in Example 1. Ammonium chloride was removed and the filtrate neutralized to pH 6.5. with Nal-ICQa. Part of the alc ho w d t ed off e en the P diw gelled. "The gelatinous material was siibl'ecteci to. st ll n a i ms ure and e ma n f i ct eu 4e- 9. 6 n e n i lw d g 'l iei 138-'140'C. e N a a F un 5 c fa 483 Refractive index (12:25"); 1.4440 Analysis agreed with the formula: H CHr Q-Q CHCOO 02115 P OfO i i Acetyl derivatiue.,One-eighth mole (36.1 g.) of thefree ester was acetylated as described in Ex: ample l andv the product distilled at 1:2 -mm. pressure. The main fraction (36 g. or 90v percent yield). distilledat 180-1 85 o.

N anal: Found'5.38; calcd. 5.44 Refractiveindex (t;25".) 1.4650.

nal si was? w t h rmu a .N- 3.-Q Q Q l7 Mi 1; M mic .4 ar a 4 r @2. i i e-.A equs he et rlrsie was re ared. n .9 m re exam es. Qi ei' a ters may be prepared by employingsome hav he ame echn e n -hal b f i '6? ih .-a a i tr a d ih 350 ml. of n-butyl alcohol and 154 g of dry HCl' is de c ibed n x mple. 'Afi j mev' i i th HiZC the fi r e a d int We" a portions.

Fr e e cster.One portion was neutralized topH 6.5 it aNaI-ICQ: and unreacted butyl'alcohol removed by evaporating on a steam bath.- The r sid e wa i t ed at. 14mm. ress-ename its. ma n rac d t l n a ;6 32 Ureqn e.- distilling. the fraction (69 percent overall yield)- distilling at 12 0,-125 C. was saved.

N anal: Found 5.19; calcd. 5.12

Refractive index (12:25 1.4334. A Analysis agreed with the formula:

Acetyl derivative-The second portion of the filtrate was neutralized with NaI-ICOa and unre. acted butyl alcohol removed by evaporating on a steam bath. The oilwas dissolvedin 0.25 mole of glacial acetic acid and 0.5 mole. of acetic anhy-L dride added. After standing overnightthe acetic acid and unreacted anhydride. was distilled 91T The oily residue was distilled at 1-2. mm. pressure an the fraction (:64 g. or 81 ge xgent 1d); distll ing atl5 6 +157 retained.

N, anaL: Found 4.39; calcd. 4.40 iv inde 65 .5 .2

Mixed ammo acid s.:A commercial sample, consisting primarily of leucine, isoleucine and eeth eii n New ercen ei d et ee a by rqd a m h m nu c ure o menes iiiei emanate. used rude rak eq g was ir t rea d with acryie i r y s spend n wate n a din s dium dro de males. e u val nt t the i im eii nt .Q mi o a d a enej u e e t. ba ed Q11. s e c nt n Qf ac y nit ile w saqd and a er. standing overnight at room temperature warmed to 65- 'Z0""C. for 2 hours. 'The'rea tion mixture was acidified with 1.366 moles of hydro: chloric acid and evaporated to dryness. The dryresidue was treated withone liter 'of 99 percent ethyl alcohol and 6.5'inoles0f dryHC-l asde scribed in Example 1. After reducing the volume by evaporating on a steam bath to remove excess HCl, the salts were filtered off. The colored solution was neutralized to pH 7 .0 .withNazCOa. and again filtered. The alcohol was distilledoff, leaving a colored oily residue weighing. 265.5-g'." (N=5.42 percent). The residue was distilledat 1-2 mm. pressure and .3 fractions: collect'd,fas

shown i-n: Table I.

Table I Fraction B.P./l2 mm, Perbcxent EXAMPLE 12 Acetyl derivative.A neutralized oily residue obtained as in the above example was acetylated as described in Example 1, and subjected to distillation at 1-2 mm. pressure. Two fractions were obtained as shown in Table II.

Table II Weight Fraction (g) 9 Percent B.P./l-2 mm. N

l 2 Residue in still I no-Qcm-on-c o-o 01m 3. N (Z-carbethoxyethyl) -leucine-ethy1-ester IOH| NH-CHz-CHr-C O-O CzHs oH-om-om-o -0 01H;

on. g I 4. N-(2-carbethoxyethyl) -diethylaspartate Niacin-on o o -o 02H:

H-C 0-0 CzH: m-oo-o 01H;

5. N acetyl, N -(2-carbethoxyethyl) -diethy1- aspartate of the following formula cm-o O-N-CHr-CHr-C 0 o 01H:

H-C o-o 01H} Hr-C O0 Cz s 6. N acctyl, N (2-carbethoxyethyl) -methionine-ethyl-ester of the following formula:

CHr-CO-N-CHa-CHz-C 0-0 02H:

CHr-S-CH;-CHr-CH-COO 01H;

7. The process which comprises subjecting N- (Z-cyanoethyl) -L-tyrosine to alcoholysis by reacting with an aliphatic alcohol in the presence of a non-oxidizing mineral acid and adding suflicient alkali to the reaction mixture to free the reluitinz ester.

8. The process which comprises subjecting N- (Z-cyanoethyl) -L-leucine to alc'oholysis by reacting with an aliphatic alcohol in the presence of a non-oxidizing mineral acid and adding sufficient alkali to the reaction mixture to free the resulting ester.

9. The process which comprises subjecting N- '(Z-cyanoethyl) -'DL-methionine to alcoholysis by reacting with an aliphatic alcohol in the presence of a non-oxidizing mineral acid and adding suffiqcicnt alkali to the reaction mixture to free the resulting ester.

10. The process which comprises subjecting N- (2-cyanoethyl) -DL-aspartic acid to alcoholysis by reacting with an aliphatic alcohol in the presence of a non-oxidizing mineral acid and adding sufiicient alkali to the reaction mixture to free the resulting ester.

11. The process which comprises subjecting a mixture of cyanoethylated amino acids obtained by reacting the amino acid cake byproduct from the manufacture of monosodium glutanate, said cake consisting primarily of leucine, isoleucine and methionine, to alcoholysis by reacting said mixture with an aliphatic alcohol in the presence of a non-oxidizing mineral acidand adding sufiicient alkali to the reaction mixture to free the resulting mixed esters.

12. Process according to claim 11 in which the resulting mixed esters are further subjected to theaction of an organic acylating agent whereby the amino hydrogens are replaced by the organic acyl'groups.

13. A process which comprises subjecting a compound of the following structural formula in which R is a member selected from the group consisting of aliphatic and araliphatic radicals, and n is an integer from 1 to 2, to simultaneous hydrolysis and esterification to convert the nitrile group and the carboxyl group into ester groups by reacting with an aliphatic alcohol in the presence of a non-oxidizing mineral acid, adding sufficient alkali to the reaction mixture to free the resulting ester, separating the free ester from the reaction mixture and subjecting it to theaction of an organic acylating agent whereby the amino hydrogen is replaced by an organic acyl group.

LEONARD L. MCKINNEY. EUGENE H. UI-IING. EUGENE A. SETZKORN. I JOHN C. COWAN.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS OTHER REFERENCES Ruzicka et al., Helv. Chim. Acta 5, pages 715-720 (1922). 

1. A NEW COMPOSITION OF MATTER OF THE GROUP CONSISTING OF N-(2 CARBETHOXYETHYL) - TYROSINEETHYL ESTER N-2-CARBETHOXYETHYL) - LEUCINE-ETHYL ESTER, N-(2CARBETHOXYETHL) - DIETHLASPARTATE, N-ACETYL, N-(2-CARBETHOYETHYL- DIETHLASPARTATE AND N-ACETYL, N-(2-CARBETHOXYETHYL) METHIONINE-ETHYL-ESTER. 